Collagen-specific enzyme with platelet aggregation inhibition properties

ABSTRACT

The collagenase is like tissue collagenases in that it cleaves collagen at a single site. The collagenase which has a molecular weight of about 50 k daltons, specifically inhibits collagen-induced platelet aggregation, with substantially no effect on platelet count or size, and may be used alone or together with another platelet aggregation inhibitor. The latter may also be a leech-derived biochemical, such as an apyrase, or an inhibitor of the release of platelet aggregation factor by leucocytes.

This is a continuation of application Ser. No. 041,016, filed Apr. 10,1987, now abandoned.

The present invention is concerned with collagen-specific enzymes withplatelet aggregation inhibition properties; for brevity we will refer tosuch enzymes herein as collagenases.

The name collagenase is used for an important group of enzymes whichspecifically degrade collagen by means of hydrolytic scissions ofpeptide bonds in the helical regions when the collagen is in the native(undenatured) state. There are basically two types of collagenases:

(a) those making multiple non-specific hydrolytic scissions of thecollagen molecule. This type of collagenase is elaborated by microorganisms that in themselves do not contain collagen;

(b) those which make a very limited number, typically one cut, percollagen molecule. This type of collagenase, the so-called tissuecollagenases, is produced by mammals and other advanced animals that infact have collagen as a major extracellular component of their tissues(Harris, E. D. and E. C. Cartwright 1977 in, Proteinases in MammalianCells and Tissues, Elsevier, pp 249-282).

Collagen is a structural protein which is virtually ubiquitousthroughout the human body and bodies of higher animals. It ischaracterized by regions of small, repeating sequences of amino acidswhich result in the formation of helical chains between molecules. Thesehelices give rise to its exceptional structural stability and strength.As a major constituent of connective tissue and skin, and theextracellular matrix (cement) which binds cells together, and as a majortrigger in the aggregation of platelets collagen is of considerablephysiological importance. An enzyme which specifically cleaves collagenshould have a wide variety of medical and scientific applications.

Collagenases are widely distributed in nature in, for example, bacteriaand vertebrate tissue. Bacterial collagenases, e.g. those produced byClostridium histolyticum, are of the first type; that is, they cleavethe collagen molecule at approximately 200 sites. This type is concernedwith invasion of a host and, through degradation of the collagenmolecule, with nutrition. Mammalian and other vertebrate collagenasesare of the second type; that is, they cleave collagen at only onespecific site. This type is concerned with repair of tissues, removinginjured collagen, and remodelling of specialized tissues at a particulartime of development or physiological expression, e.g. resorption of thetadpole tail or involution of the postpartum uterus. Followingbiochemical extraction and purification, tissue collagenases occur inlatent form which requires specific activation. Harris, E. D. and E. C.Cartwright 1977, in, Proteinases in Mammalian cells and tissues,Elsevier, pp 249-282; Cawston, T. E. and G. Murphy 1981. Methods Enzymol80, pp 711-734. This property severely limits the commercial usefulnessof the known tissue collagenases. In fact relatively little is knownabout tissue collagenase because of the technical difficulties of havingactive enzyme in pure form.

This invention is concerned with a new type of tissue collagenase whichoccurs in active form following extraction. This active type ofcollagenase can be derived from the cephalic tissue of leeches. We havenow isolated this new type of tissue collagenase from a variety ofarhynchobdellid and rhynchobdellid leech species, including members ofthe family Hirudinidae, e.g. Hirudo medicinalis, Hirudinaria manillensisand Poecilobdella granulosa; the family Haemadipsidae e.g. Haemadipsazeylanica and Haemadipsa picta; and the family Glossiphoniidae, e.g.Haementeria ghilianii (all the above being as defined in "Leech Biologyand Behaviour" by Dr. R. T. Sawyer, Oxford University Press, 1986).

According to the invention, therefore, there is provided a collagenasewhich is such that it cleaves native (undenatured) collagen at a singlesite, said collagenase being characterised by the fact that it is leechderived.

The leech collagenase according to the invention belongs to the tissuetype of collagenases in that it cleaves at a single primary site of thecollagen molecule. By way of example, the action of leech collagenase onType I collagen from calf skin is revealed by SDS-PAGE to result in theprogressive cleavage of α chains into large α^(A) and small α^(B)fragments (FIG. 1). The same example demonstrates that leechcollagenase, unlike tissue collagenases from mammals and othervertebrates, occurs in active form.

The leech collagenase according to the invention inhibits thecollagen-induced aggregation of platelets (or thrombocytes) in vitro,while having substantially no effect on either platelet count orplatelet size. This activity is highly specific, and is not due to someindirect effect but due to a direct effect on the induction of plateletaggregation by collagen. (Bacterial collagenase, in contrast, does notinhibit platelet aggregation).

It is known that collagen is an activator of two mechanisms of plateletaggregation. In a first mechanism, known as the arachidonic acidpathway, collagen stimulates phospholipase A₂ within the platelets; thisreaction in turn releases arachidonic acid which is converted throughthe action of cyclo-oxygenase enzyme to thromboxane A₂. The lattermobilises intraplatelet calcium and causes release from the plateletgranules of other proteins, such as serotonin and platelet derivedgrowth factor, and also releases nucleotides such as adenosinediphosphate (ADP).

In a second mechanism, subendothelial collagen and thrombin activate theplatelets, granule release of calcium occurs, and also release ofplatelet derived growth factor 4, and the platelets adhere directly tothe subendothelium.

The action of the leech collagenase according to the invention is toinhibit platelet aggregation which proceeds along either of these twomechanisms.

The leech-derived collagenase according to the invention is furthercharacterized by the following properties:

(a) It comprises a polypeptide of molecular weight 50,000±5,000, whenmeasured by gel chromatography using Fractogel TSK HW (55F) (FIG. 2);

(b) It appears to retain significant activity after solvent extractionwhereas mammalian-type collagenase does not;

(c) It readily digests native collagen and denatured collagen (gelatin).

The leech collagenase according to the invention has a large number ofpotential uses, for example, as follows:

(a) as a research biochemical, for example for the study of nativecollagen and its relationship to other extracellular components, and theidentification, characterisation and quantification of collagen;

(b) for use in tissue culture, for example, for dissociating tissue,such as leech collagenase-digested cells for the production of vaccinesand the like;

(c) as a research tool for inhibiting platelet aggregation in vivo, foruse in studying the mechanism of collagen-induced platelet aggregationand thromboses;

(d) in the treatment of hide, fur, meat (for example, to soften hide orfur, tenderize meat or treat sausage casings or the like).

(e) for recovery of silver from photographic waste; and

(f) for use in therapy.

In connection with use in therapy, the leech collagenase according tothe invention may have the following uses:

1. selective degradation of collagen in the eye to remove scar tissue(for example, after cataract surgery, retinal detachment and pars planavitretomy);

2. removal of necrotic tissue produced by ulcers or burns and to inducegrowth of normal tissue;

3. treatment and prevention of cicatrices (such as acne scars, keloids,wrinkles and cellulitis);

4. enhancement of penetration of a pharmaceutically active materialthrough the skin (optionally together with leech-derived hyalurodinaseas described in European Patent Specification 193330);

5. treatment of dental transplants and root canals, for removal ofodontoblasts for example;

6. treatment of herniated intervertabral discs by injection;

7. stimulation or inhibition of angiogenesis;

8. treatment of Peyronie's disease by administration of the collagenasedirectly on to the plaques which form in the course of the disease; and

9. inhibition of platelet aggregation in vivo.

In connection with the latter, many synthetically produced chemicalsused as inhibitors of platelet aggregation (such as dipyridamole,aspirin, sulphinpyrazone, ticlopidine, prostacyclin and thromboxanesynthetase inhibitor) are non-specific and affect many metabolicprocesses in addition to platelet aggregation. The leech collagenaseaccording to the invention has less undesirable side-effects (presumablybecause it has evolved biologically to do as little damage to the host'sbody as possible) and acts directly on one of the fundamental inducersof platelet aggregation.

The use of collagenase for the inhibition of platelet aggregation (invivo or in vitro) is a very important aspect of the present invention.As far as we are aware, no-one has previously suggested the use oftissue-type collagenase (that is collagenase which cleaves collagen at asingle site) for this purpose and according to a further aspect of thepresent invention, therefore, there is provided collagenase whichcleaves collagen at a single site for use in the inhibition of plateletaggregation.

In this connection, the collagenase may be used (alone or together withother active factors) for the following purposes.

(i) in micro-surgery to induce prolonged localised bleeding and torestore blood circulation in congested implants or skin or tissue flaps;

(ii) as a prophylactic treatment of transplanted tissue, such as veinsused in coronary by-pass surgery, to prevent collagen-associatedplatelet aggregation;

(iii) to reduce the risk of post-surgical clots by denaturing rawcollagen exposed to blood, by treatment of collagenase at the site ofsurgery and by circulation of collagen in the blood to inactivateexposed collagen;

(iv) to reduce aggregation of platelets along atheromatous plaques andalong blood vessel fractures where collagen is exposed;

(v) to reduce the risk of platelet aggregation whenever acollagen-associated prosthesis or implant is employed.

The leech collagenase according to the invention may be employed insubstantially pure form, or together with one or more further inhibitorsof platelet aggregation (which inhibit platelet aggregation induced bythe same or a different factor). It is sometimes desirable to employ theleech collagenase in conjunction with an inhibitor for other mechanismsof platelet aggregation. Several such inhibitors (believed to be novelper se) have been found to be present in leech saliva. Indeed, at leastthree such inhibitors have been found in leech saliva, one being anapyrase (an ADP-induced platelet aggregation inhibitor) of molecularweight approximately 400,000, a second being an apyrase of molecularweight approximately 45,000, and a third being an inhibitor of therelease of platelet aggregation factor by leucocytes and having amolecular weight of about 600. The leech collagenase according to theinvention may be used together with one or more such inhibitors.

In order that the present invention may be more fully understood thefollowing Examples are given by way of illustration only.

EXAMPLE 1

Crude extract containing leech collagenase was collected from starvedHirudo medicinalis in the following manner. The leeches were allowed tofeed through a dried calf intestine membrane on a solution of 0.1 mMarginine in saline. The ingested leech was then made to regurgitate, theregurgitant constituting the crude extract. Apart from collagenase theextract contained no other detectable proteases. This is in sharpcontrast to the micro-organism derived collagenase, which was heavilycontaminated with proteolytic enzymes.

The crude extract was fractionated with 0.2M ammonium bicarbonate on a2×90 cm Fractogel TSK HW (55 F) column. The eluate was collected at 2 mlintervals at a flow rate of 41 ml per hour.

The fractions were assayed by incubating leech fraction with ¹²⁵I-collagen-sepharose in 0.05% bovine serum albumin/5 mM CaCl₂ /75 mMHEPES buffer, pH 7.5 for 5 hours at 25° C. Activity was measured byrelease of ¹²⁵ I from the Sepharose-bound collagen. One collagenase unit(CU) is that amount which degrades 1 μg of iodinated collagen-sepharose.The assay is linear over 5 hours.

Leech collagenase was found in peak fraction 115 (FIG. 2a). Bycomparison with markers of known molecular weight, the molecular weightof leech collagenase is taken to be approximately 50,000±5,000 daltons(FIG. 2b). Similar results were found with collagenase extracted fromPoecilobdella granulosa.

EXAMPLE 2

The mechanism of cleavage of leech collagenase was determined asfollows. Crude extract was prepared as per Example 1. The crude extract(A₂₈₀ 0.077) was incubated with Type I collagen (1 mg ml⁻¹) from calfskin in 1.5 mM CaCl₂ /10 mM Tris-HCl buffer pH 7.8 (total volume 100 μl)at 37° C. for 1,5 and 24 hours. Collagen alone and clostridiumcollagenase (0.1 mg ml⁻¹) were used as controls. Solubilizing mixture(120 μl, Nature 227, p. 680) was then added and the mixtures were placedin a water bath for 3 minutes. 50 microliters of mixture were placed ineach well of SDS-PAGE gel.

The results are presented in FIG. 1. Lanes A and F, collagen controls;B, C and D, crude extract incubated for 1, 5 and 24 hours respectively;E, clostridium collagenase incubated for 1 hour; G, molecular weightmarkers.

It may be seen that the isolated α chains are progressively cleaved intolarge α^(A) fragments and small α^(B) fragments. This highly specificcleavage pattern characterizes leech collagenase as a tissue typecollagenase, and is in sharp contrast to the non-specific cleavage bycollagenases from micro organisms. It can also be seen that, in contrastto the latent form of tissue collagenases from mammals, leechcollagenase occurs in active form in the crude extract.

EXAMPLE 3

A sensitive assay for measuring tissue-type collagenase has been devisedinvolving detection of collagenase inhibition of collagen-inducedplatelet aggregation.

(a) Tissue type collagenase (Human synovial fibroblast collagenase) (3collagenase units or CU) and clostridium collagenase (50 μg, 12 CU) wereexamined for their effects on platelet aggregation. Tissue-typecollagenase greatly reduced collagen-induced platelet aggregationwhereas clostridium collagenase, at a four-fold concentration ofcollagenase units in the test mixture, was practically without effect.

The above demonstrates that the above assay is specific for tissue typecollagenases.

(b) Crude leech extract was prepared and assayed as per Example 1.Apyrase was removed from the crude extract by passing it through a 0.2ml GTP-agarose column washed with 1 mM arginine/saline. The apyrase-freeextract strongly inhibits platelet aggregation induced by collagen. Themeasured leech collagenase activity was 2.25 collagenase units.

The above demonstrates that leech collagenase specifically inhibitscollagen-induced platelet aggregation. In the above examples (a) and (b)Platelet Rich Plasma (PRP) (400 μl) HEPES buffer pH 7.35, and the testsolution were pre-incubated for 1 minute, when 1.2 μg/ml collagen wasadded (final reaction volume 500 μl). Aggregation was followed by theincrease in light transmission against time, as recorded on anaggregometer chart. A₂₈₀ values of the crude extract in the testmixtures (×10³) were evaluated as platelet aggregometer tracings. Themeasured collagenase activity was 15.3 CU per 1.0 A₂₈₀.

(c) Crude leech extract (Stage I) from cephalic tissue of buffaloleeches was fractionated as follows. The head regions of 40 buffaloleeches Poecilobdella granulosa were removed and weighed (32.8 g freshweight). They were homogenized in distilled water for ten minutes at 4°C. The suspension was centrifuged at 650 g for 10 minutes at 4° C. Thesupernatant is saved, while the precipitate is resuspended andcentrifuged again. The supernatants are combined to give Stage I enzyme.

The effect of the isolated proteinacous material on platelet aggregationhas been studied in vitro using platelet-rich plasma (PRP) produced bycentrifuging fresh blood at 800 g for 5 minutes. The PRP produced waspreincubated with isotonic leech extract of varying stages of purity.Controls were performed in which isotonic saline replaced the leechextract. After this pre-incubation the PRP was incubated with a varietyof inducers of platelet aggregation, namely collagen, ADP andristocetin.

Results are obtained using an EEL platelet aggregometer by measurementof the optical density of the incubated mixture with time, the mixturebecoming less turbid as platelet aggregation occurs. A blank preparationof platelet-poor plasma was used to give an optical density value for asolution lacking platelets.

Stage I was fractionated on G75 Sephadex gel (1.6×75 cm). Elution wasperformed using 50 mM-Tris HCl, 20 mM NaCl, pH 7.0 at a rate of 1.0 mlper minute. Fractions were collected at 5 minute intervals and theirprotein content measured on a spectrophotometer by absorbance at 280 nm.Protein was eluted with peaks measured in Fractions 4 and 26. Each ofthe fractions collected, as well as the crude extract, were examined forinhibition of collagen-induced platelet aggregation as described above.Only fractions 4 and 26 as well as crude extract were found to containactivity capable of substantially inhibiting aggregation induced by 1 μgml⁻² collagen. Fraction 26 contained collagenase activity. Fraction 4contained apyrase activity (10 μg ml⁻¹ ADP).

The crude extract in serial dilution was found to inhibit significantlycollagen induced platelet aggregation induced by 1 μg ml⁻¹ collagen at0.1, 0.01 and 0.001 dilution.

The crude extract itself extensively inhibited platelet aggegation evenat concentrations of collagen of 40 μg ml⁻¹ which is ten times higherthan that normally used to induce platelet aggregation in vitro.Ristocetin-induced platelet aggregation was not inhibited bypre-incubation with leech extract according to the invention.Furthermore, leech extract does not affect platelet count nor plateletsize. Also our finding that ADP-induced platelet aggregation is nottotally blocked with leech apyrase or collagenase indicates anotherinhibitor of platelet aggregation is present in the leech.

The enzyme was further purified in the following manner. To the 40%supernatant containing the enzyme ammonium sulphate was added at 80%saturation. The suspension was centrifuged at 800 g for 20 minutes at 4°C. The pellet is resuspended in buffered solution and dialysed threetimes against distilled water at 4° C.

The resuspended 80% pellet was assayed by the sensitive techniquedescribed in Example 3 in which collagenase inhibits collagen-inducedplatelet aggregation. Platelet aggregation induced by collagen was foundto be extensively inhibited by the 80% pellet.

The 80% supernatant had significantly reduced ability to inhibitcollagen-induced platelet aggregation.

When assayed for collagenase activity, as per Example 4, the 80% pelletcontained collagenase activity whereas the 80% supernatant did not.

That the 80% supernatant had some platelet inhibition but no collagenasemight be interpreted as evidence for the presence of a low molecularweight inhibition of platelet aggregation in the 80% fraction, notcollagenase. Further evidence is presented by the inhibition by the 80%supernatant but not the 80% pellet, of the secondary wave of aggregationin the presence of high (100 mg ml⁻¹) and medium (50 mg ml⁻¹)concentrations of ADP.

EXAMPLE 4

Leech collagenase was further characterized as follows:

The frozen head regions of buffalo leeches Poecilobdella granulosa wereweighed (114 g fresh weight). The tissue was homogenized in distilledwater for 10 minutes at 4° C. The suspension was centrifuged at 650 gfor 10 minutes at 4° C. The supernatant was saved, while the precipitatewas resuspended and centrifuged again. The supernatants were combined togive Stage I enzyme, total volume 146 ml. The enzyme was furtherpurified by adding 40% saturated ammonium sulphate to Stage Isupernatant. The suspension was centrifuged at 800 g for 20 minutes at4° C. To the 40% supernatant was added 80% saturated ammonium sulphate.The suspension was centrifuged as before. The 80% pellet was suspended,to which was added 50% ammonium sulphate. Following dialysis againstdistilled water at 4° C., the Stage I enzyme, 40%, 80% and 50% pelletsand supernatants were assayed for total protein and collagenaseactivity, respectively. Boiled Stage I served as control.

The fractions were assayed for collagenase activity by incubating 100 μlof each fraction in 100 μl of collagen (2% gelatin) for 24 hours. Afterincubation the solutions were placed onto ice for 30 minutes. Failure ofthe solution to gel upon cooling results from collagenase activity.

Results were as follows.

    ______________________________________                                                   Collagenase Activity                                                          (% gelatine digested)                                                                        Protein                                             ______________________________________                                        Stage I      100%             24     mg/ml                                    40% pellet   100%             12.6   mg/ml                                    40% supernatant                                                                             50%             7      mg/ml                                    80% pellet   100%             8      mg/ml                                    80% supernatant                                                                             0%              0.12   mg/ml                                    50% pellet    50%             16     mg/ml                                    50% supernatant                                                                             0%              2.6    mg/ml                                    ______________________________________                                    

It can be seen from the above experiment that leech collagenase is amolecule which is precipitated by both 80% ammonium sulphate and 50%ammonium sulphate. It can be dissolved in part in 40% ammonium sulphate.

A two ml sample of 50% ammonium sulphate pellet was extensively dialysedagainst 5 liters distilled water, made up to 1M-NaCl and then applied toa G75 superfine Sephadex column that had been equilibrated with 50mM-Tris HCl, 20 mM-NaCl pH 7.5. The gel bed was 70 cm high and 1.6 cm indiameter with a flow rate of 20 ml per hour. Fractions were collected at20 min. intervals.

An estimate of the protein content of each fraction was obtained bymeasurement of its absorbance at 280 nm. Measurement of fractions 0 to20 by coomassie blue dye showed the A₂₈₀ peak between fractions 7 and 14to consist of two peaks having maxima at fractions 9 and 11. (See FIG.3).

Collangenase activity was assayed by incubation of 100 μl of eachfraction with 100 μl 4% gelatin at 37° C. for 3 hours. The samples werethen kept on ice for 10 minutes and the degree of liquefaction of themixture observed by inversion. Controls containing buffer instead ofcollected fractions were prepared and shown to exhibit no liquefaction.Aliquots of each fraction off the column were also heated at 100° C. for10 minutes and then assayed in a similar manner for collagenase. Resultsare shown in FIG. 4. It can be seen that the collagenase activitycorresponds with the first protein peak at fractions 8 and 9 and alsowith the proteins of lower molecular weight between fractions 20 and 40.

Measurement of the ability of each fraction to inhibit collagen-inducedplatelet aggregation was also studied. Using a platelet aggregometer,the extent of inhibition was studied by adding 50 μl of 4 μg per mlcollagen to a mixture of 100 μl fraction and 400 μl platelet rich plasma(300±50×10³ per cu mm). Aggregation in each case was expressed as apercentage of the aggregation induced by a similar amount of collagenwhen the fraction was replaced by a similar volume of isotonic saline. Across section of the results obtained are shown in FIG. 4. Fractions 8and 9 showed complete inhibition of collagen-induced activity. All otherfractions showed no significant effect upon collagen-induced plateletaggregation.

Similar results were found with enzyme extracted from Hirudomedicinalis.

EXAMPLE 5

That leech collagenase occurs in rhynchobdellid leeches is demonstratedby the following example on the glossi-phoniid Haementeria ghilianii.

The entire salivary gland/proboscis complex was dissected from twomedium-sized (5 cm) individuals of Haementeria ghiliannii. The frozenglands were homogenized with a mortar and pestle to which was added 1.5ml of distilled water. The total extract was centrifuged for one minute.From both the supernatant and pellet 100 μl samples were added to wellscontaining 100 μl of 3% gelatin. The solutions were allowed to digestfor 24 hours at 22° C. Collagenase activity, i.e. total digestions ofthe gelatine, occured only in the pellet. The supernatant had nocollagenase activity.

EXAMPLE 6

The pharmaceutical potential of leech collagenase as inhibitor ofplatelet aggregation is demonstrated by the following example:

An adult medicinal leech Hirudo medicinalis (2.6 g) was allowed to feedto repletion on the forearm of a human subject. Blood and otherparameters were carefully monitored before, during and after theexperiment. Baseline blood parameters from a lanced thumb before feedingserved as baseline control: clotting occurred in 31/2 minutes, andplatelet aggregation was clearly visible on smears under the microscopein about half that time.

The leech fed for 58 minutes during which time it sucked 4.5 ml ofblood. The wound continued to bleed for more than 3 hours, at a flowrate of 150 μl/min. Blood coming from the wound was sampled at frequentintervals. Clotting time was found to be as follows:

    ______________________________________                                        Time Following Bite                                                                            Clotting Time                                                ______________________________________                                         0 mins          11 mins                                                       5 mins           7 mins                                                      10 mins          31/2 mins                                                    20 mins          31/2 mins                                                    ______________________________________                                    

These and related data prove that contrary to current views theprolonged bleeding following a leech bite is not due to nirudin (leechantithrombin); with respect to ability to clot (fibrin formation) theblood returns to normal within 10 minutes, but bleeding persists.

Our research shows that the prolonged localised bleeding is due toinhibition of platelet aggregation. In the example when the platelets inthe fresh blood were examined by several means, including blood smearson glass slides and a canalyzee which counts platelets; it was foundthat the platelets showed no tendency to aggregate for more than anhour. With a baseline value of 203,000 platelets per ml of blood, theplatelet number was as follows:

    ______________________________________                                        Time Following Bite                                                                            Platelet Number                                              ______________________________________                                        11/2 mins        201,000                                                      20 mins          202,000                                                      60 mins          200,000                                                      150 mins         195,000                                                      ______________________________________                                    

At least after one hour the platelets themselves appear to be normal asdemonstrated by the representative observation, at 150 min, that bloodseveral minutes old which had come into contact with skin and old bloodhad a platelet count of 96,000, a reduction indicative of plateletclumping/aggregation.

Prolonged localized bleeding following a leech bite is best interpreted,in the light of Examples 1-3, as follows. Leech collagenase secreted inthe saliva during biting and feeding modifies exposed raw collagensurrounding the damaged vessel wall in such a way as to make itincapable of stimulating aggregation of platelets in passing blood.Collagen is the most potent inducer of platelet aggregation.

The inhibition of platelet aggegation by leech collagenase as describedabove would be therapeutically very useful, for example in micro-surgeryto restore circulation wherever blood is congested such as followingsurgical implants and tissue flaps and in haematomas.

We claim:
 1. A substantially pure collagenase isolated from leech tissueor from leech secretion, said collagenase being capable of (a) causinghydrolytic scission of peptide bonds in the helical regions ofundenatured collagen molecules, (b) causing only one cut per chain ofsaid collagen molecules, and (c) digesting gelatin.
 2. The collagenaseof claim 1 wherein the collagenase is also capable of inhibitingcollagen-induced platelet aggregation.
 3. The collagenase of claim 2wherein the molecular weight is from 45 to 55 kdaltons.
 4. A method forisolating collagenase according to claim 1, which method comprises thesteps of(a) fractionating a fluid regurgitated by leeches by means of agel fractionation; and (b) collecting eluate fractions containing saidcollagenase.